Modulators of cysteine protease

ABSTRACT

The present invention concerns novel mediators of the activity of picornavirus 3C protease and the modulation of the activity of other similar proteins. The modulators may be used in pharmaceutical compositions either for inhibition of 3C protease (for example in viral infections) or for the enhancement of the activity of proteins similar to the 3C protease such as Apopain (for induction of apoptosis).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/IL98/00602, filed Dec. 14, 1998, which claims priority to IsraelPatent No. 122591, filed Dec. 14, 1997.

FIELD OF THE INVENTION

The present invention concerns novel pharmaceutical compositions. Inparticular, the pharmaceutical compositions of the invention comprisecysteine protease modulators. The pharmaceutical compositions of theinvention are preferably used for the treatment of viral infections, anddiseases resulting from inappropriate apoptosis.

BACKGROUND OF THE INVENTION

Cysteine proteases are a major family of peptide-bond-cleavinghydrolases, defined as proteases in which the thiol group of a cysteineeresidue serves as nucleophile in a catalysis. All known cysteineepeptidases require a second residue—an adjacent histidine—for catalysis.While the role of the histidine has been postulated to be a general basein analogy to well-understood serine proteases, it has been clearlydemonstrated in theoretical studies that the catalytic histidine cannotact as a base, rather that it acts by donating a proton to thesubstrate. Cysteinee proteases have been found in the previousliterature in viruses, bacterial protozoa, plants, mammals and fungi.

There are currently known 38 families of cysteinee proteases (C1-C38),most of which are divided into 5 separately evolved clans (CA-CE). ClanCB enzymes are chymotrypsin-like cysteine proteases containing a His/Cysdiad (catalytic histidine preceding catalytic cysteinee in the linearsequence), and responsible for proteolytic cleavage of pol polyproteins(containing the RNA polymerase). These enzymes, commonly hydrolyseglutaminyl bonds, and act on crucial cell proteins as additionalsubstrates.

Peptidases of Clan CA include vital mammalian enzymes such as papain orcathepsins. The normal activity of these enzymes is essential and theiractivity should not be inhibited by any type of pharmaceuticalcomposition.

Clan CC includes sixteen (16) families of papain-like viral peptidases(C6-C9,C16,C21,C23,C27-29,C31-C36), comprising a cys/his diad. Despitesequences similar to Clan CA enzymes, these viral proteins arefunctionally similar to Clan CB enzymes, which cleave viralpolyproteins.

Clan CD is represented by a single family (C14), which comprisescytosolic endopeptidases found only in animals. Cytosolic endopeptidasesare involved in the process of apoptosis (programmed cell death).

There is no structural data regarding peptidases of Clan CE (family C5adenovirus endopeptidase), as well as untyped enzymes. One untypedfamily, however, C13, which includes medically important proteases suchas Schistosoma mansoni haeomoglobinase, is similar to the substratespecificity of Clan CB enzymes (asparaginyl compared to glutaminylbonds). Additionally, C13 has a low sensitivity to E64. Interestingly,this latter property may indicate a fold similar to Clan CBchymotrypsin-like-enzymes.

Picornaviruses are single-stranded positive RNA viruses that areencapsulated in a protein capsid. These viruses cause a wide range ofdiseases in man and animal including common cold, poliomyelitis,hepatitis A, encephalitis, meningitis and foot-and-mouth disease, aswell as diseases in plants such as the potty disease in potatoes. Afterinclusion into the host cell, the picornaviral RNA is translated into a247-kDa protein that is co- and post-translationally cleaved yieldingeleven (11) mature proteins. Cysteine proteases denoted 2A and 3C, whichare part of the picornaviral self polyprotein are responsible for thesecleavages. The 2A protease cleaves co-translationally between thestructural and non-structural proteins and the 3C protease cleavespost-translationally the remaining cleavage sites except one.

Having been recognized as important proteins in the maturation of thepicornaviral life cycle the 3C and 2A proteases have been a prime targetfor extensive structural and mechanistic investigations during the lastfew years. Recently, their mechanism and structural features have beendetermined (Kreisberg et al, Organic Reactivity: Physical and BiologicalAspects, 110-122 (1995)).

Site-directed mutagenesis studies (Cheah K. C. et al, J. Biol. Chem.,265 (13):7187-7189 (1990)) confirmed by X-ray studies (Matthews et al,Cell, 77:761-771, (1994)) led to the finding that the catalytic site of3C is composed of the following amino acids: Cys in position 146,Glu/Asp in position 71 and His in position 40. These three amino acidsin the catalytic site of the 3C enzyme constitute a hybrid between theamino acids at the catalytic site of cysteine proteases and serineproteases.

The 3C protease has been shown by mutagenesis and crystallography todepend on a his/cys diad (His40, Cys146—rhinovirus numbering). A thirdconserved residue in the 3C protease, Asp 71, was initially consideredanalogous to Asn175 (the third member in the catalytic triad of papain),however crystallography has shown this residue to be of minor catalyticimportance.

Due to the involvement of various cysteine proteases in many disordersand diseases ranging from microorganism infection (viral and bacterial)to inflammatory and tumor processes, there have been recently manyattempts to find inhibitors for cysteine proteases (Otto andSchimeister, Chem. Rev., 97:133-171, 1997)).

There have also been attempts to find suitable inhibitors of thepicornavirus 3C and 2A proteases in order to treat viral infections. Byinhibiting these proteases, the production of new virions can be avoidedbecause there are no native cellular proteases that can replace thecleavage activity of the viral proteases. Therefore, finding anefficient inhibitor against 3C and/or 2A picornavirus proteases willlead to the production of an anti-viral pharmaceutical compositionagainst a large number of viral diseases occurring both in man and inanimal.

The first agent found as an inhibitor of the 3C protease is Thysanone,an antibiotic compound obtained from Thysanophora peniciloides (Singh etal, Tetrahedron Lett., 32:5279-82 (1991)). However, this compound wasnot developed into a pharmaceutical composition because it was found tobe an efficient inhibitor of the enzyme elastase present inerythrocytes.

Two additional antibiotic compounds of fungal origin, citrinin hydrateand radicinin, were obtained by screening microbial extracts (Kadam etal, J. Antibiotics 7:836-839 (1994)). These novel two compounds showed alower level of inhibition than thysanone. The same year a new compoundtermed kalafungin, which is also an antibiotic compound, was discoveredby structural comparison to radicinin. Kalafungin was found to be abetter inhibitor (by three orders of magnitude) than radicinin andcitrinin hydrate (McCall et al, Biotechology, 12:1012-1016 (1994)).

Another group of inhibitors, substituted isatins, has also been examined(S. E. Webber, et al., Med. Chem., 39:5072-5082, 1996). Certain membersof this group show significant inhibition of 3C proteases withconcentrations in the nanomolar range, but are highly toxic. Othermembers of the group are relatively non-toxic, but have poor antiviralactivity. It has recently been shown that peptidyl Michael acceptorsinhibit rhinovirus replication at low micromolar concentrations with atherapeutic index exceeding ten (10) (Kong et al., J. Med. Chem.,41:2579-2587 (1998). Rhinovirus inhibition has also been accomplished atnanomolar concentrations at peptidyl Michael acceptors (Dragovich etal., J. Med. Chem., 41:2819-2834 (1998)). Thus, none of the aboveinhibitors has been demonstrated to be clinically useful possessing asufficiently high therapeutic index with favorable toxicology andbioavailability profiles.

Transition-state analogs are well established as enzyme and proteaseinhibitors (Barrett, A. J. and Salvesen, G., Proteinase Inhibitors,Elsevier, 1986). Functional groups such as ketone, aldehyde,chloromethyl-ketone (REVS) and recently isatin are widely used for theinhibition of serine and cysteine proteases. Class-specificity isachieved by utilization of phosphine or boron geometries (serineproteases) or groups such as epoxide (Albeck, M., Fluss, S. and Persky,R., J. Am. Chem. Soc., 118:3591-3596, 1996), cyclopropenone (Ando, R.and Morinaka, Y., J. Am. Chem. Soc., 115:1174-1175, 1993) andvinyl-sulfones (Bromme, D., et al., Biochem, J., 315:85-89, 1996).

This approach, protease inhibition through transition-state mimicry,yields highly potent inhibitors when combined with target-specificamino-acid residues or their peptidomimetic equivalent. Unfortunately,the high molecular weight and complexity of potent TS-analogs frequentlycause transport problems, which result in diminished in vivo efficacy.

In other diseases it is desired to activate cysteine proteases. Thesediseases are characterized by deficient apoptosis, i.e. by insufficientprogrammed cell death. These diseases include certain types of cancer,viral diseases and certain autoimmune diseases.

One of the key apoptotic elements identified is Apopain (caspase-3)(Nicholson, Nature Biotech., 14:297-301 (1996)). Modulators of thisprotein are sought for the modulation of apoptosis and the provision ofnovel therapeutics, Inhibitors for Apopain are useful for the treatmentof diseases in which excessive apoptosis occurs, includingneurodegenerative diseases such as Alzheimer, Parkinson and Huntingtonand cardiovascular diseases such as ischemic cardiac damage. Enhancersof this protein are useful for the treatment of diseases in whichinsufficient apoptosis occurs, such as cancer, viral infections andcertain autoimmune diseases.

Compounds such as those discussed in WPI abstract 021538, JP abstract03271261, EP application 0244363, DE application 4126543, FR application2482859, and certain references in the Merck Index have been identifiedto treat certain diseases discussed above. However, such compounds donot treat such diseases by reacting with certain 3C protease or 3Cprotease-like proteins, which are essential to viral replication and theactivity of various proteins. Thus, it would be highly desirable toconstruct protease modulators in particular, cysteine proteasemodulators that can be administered for various pharmaceutical andmedicinal purposes to a subject.

SUMMARY OF THE INVENTION

The present invention concerns modulators of cysteine proteases and morespecifically modulators of picornavirus 3C-cysteine protease and ofsimilar proteins.

The present invention is based on the finding that several chemicalcompounds are capable of inhibiting the picornavirus 3C-cysteineprotease.

Thus, according to the first aspect of the invention termed “theinhibiting aspect” there are provided inhibitors of picornavirus 3Cproteases and inhibitors of proteins having similar activity.

The present invention is based on further findings discovered by x-rayanalysis that there exists a structural similarity between Apopain andthe rhinovirus 3C protease. Similarity in active site and catalyticmachinery between the two enzymes has suggested similar mechanism andactivity. Thus, compounds which enhance or inhibit 3C protease areassumed to have activity also towards Apopain.

Thus by a second aspect termed “the enhancing aspect”, the presentinvention concerns enhancers of 3C-like proteases such as Apopain.

In one aspect, the invention is a method for the modulation of acysteine protease target comprising exposing the target to a chemicalcomposition having a core structure

wherein

A and A′ together form a C₆ aromatic or C₅-C₇ aliphatic ring, and R₁ ishydrogen or a hydrocarbon moiety of 1 to 10 carbons which is optionallysubstituted, wherein the target has an active site and catalyticmechanism similar to apopain and rhinovirus 3C protease

In another aspect, the invention is a method for the modulation of acysteine protease target comprising exposing the target to a chemicalcomposition having an orthohydroxy keto aryl core structure

wherein

R₁ is hydrogen, a hydrocarbon moiety of 1 to 10 carbons optionallysubstituted with an aryl, an amino optionally substituted with C₁-C₂arylalkyl or C₈ alkyl, an aryl optionally substituted with hydroxyl orketo, methoxy, C₂ arylalkyl optionally substituted with hydroxyl;

T₁, T₂, T₃, and T₄ are selected from the group consisting of C, O, N orS;

Z′ is hydrogen, hydroxyl, C₁-C₄ alkoxy or —OCH₂CONH₂;

Y is hydrogen, halogen, hydroxyl, nitro, cyano, methyl or —COOCH₂CH₃with the proviso that when R₁ is a lower alkyl or arylalkyl, Z′ ishydrogen or C₁-C₄ alkoxy and T₁, T₂, T₃, and T₄ are all C, Y cannot behydrogen or alkyl;

Y′ is hydrogen, halogen, hydroxyl, nitro, methyl or methoxy;

R₃ is hydrogen, hydroxyl, methyl or C₁-C₃ alkoxy;

alternatively R₁ together with Z′, Y′ together with R₃, R₃ together withY, or Z′ together with Y′ can form an aromatic or aliphatic ringstructure optionally heterocyclic, optionally substituted with hydroxyl,keto, C₁-C₄ alkanoyl, carboxyl, alkyloxycarbonyl, or phenyl optionallysubstituted with hydroxyl,

wherein the target has an active site and catalytic mechanism similar toapopain and rhinovirus 3C protease.

In another aspect, the invention is a method for the modulation of acysteine protease target comprising exposing the target to a chemicalcomposition having an orthohydroxy keto aryl core structure

wherein

R₁ is selected from the group consisting of:

(i) hydrogen or a hydrocarbon chain from 1 to about 10 carbons longselected from the group consisting of saturated, unsaturated andfluorinated, wherein the hydrocarbon chain is unsubstituted orsubstituted with at least one R¹¹, wherein R¹¹ is selected from thegroup consisting of:

(ia) C₁-C₄ alkyl, C₂-C₄ alkenyl, C₃-C₈ cycloalkyl, C₁-C₃ alkoxy or arylwhich may be unsubstituted or substituted with halogen, hydroxy, methyl,ethyl, acetyl, carboxamide, nitro, sulfamide, phenyl or sulfamyl;

(ib) halogen, cyano, nitro, amino, hydroxy, adamantyl, carbamyl,carbamyloxy or keto;

(ic) an oligopeptide of 1-4 amino acid residues, and

(id) NR¹³R¹⁴, CO₂R¹³, O(C═OR¹³), SO₂R¹³, SOR¹³R¹⁴, (C═O)NR¹³R¹⁴, orNR¹⁴(C═O)R¹³;

wherein:

R¹³ is selected from the group consisting of hydrogen, phenyl, benzyl,C₁-C₆ alkyl and C₃-C₆ cycloalkyl; and

R¹⁴ is selected from the group consisting of hydrogen, hydroxyl, andbenzyl;

(ii) an oligopeptide of 1 to 5 amino acids;

(iii) C₃-C₆ cycloalkyl, C₆-C₁₀ bicycloalkyl, C₃-C₇ cycloalkylmethyl, orC₇-C₁₀ arylalkyl, which may be additionally substituted with R¹¹ asdefined above; and

(iv) C₁-C₅ alkoxy optionally substituted with 1-3 R¹¹, NH-W or NW₂,wherein W is a substituent as defined in (i), (ii) or (iii) above;

T₁, T₂, T₃, and T₄ are selected from the group consisting of C, O, N orS;

Z′ is hydrogen, hydroxyl or C₁-C₄ alkoxy optionally containing 1-2unsaturations and substituted with 1-3 R¹⁵), or C₅-C₇ carbocyclic orheterocyclic ring system connected to R₁ optionally containing 1-2unsaturations, wherein R¹⁵ is selected from phenyl optionallysubstituted with 1-3 R¹⁴, naphthyl optionally substituted with 1-3 R¹⁴,or a C₃-C₆ heterocyclic ring system fused to an aromatic ring optionallycontaining 1-2 nonbenzenoid unsaturations and optionally substitutedwith 1-3 R¹⁴;

Y and Y′ are independently selected from the group consisting of:

(i) hydrogen, hydroxyl, halogen, C₁-C₄ haloalkyl, C₁-C₄ haloalkoxy; orC₁-C₃ alkyl which may be additionally substituted with 1-3 R¹¹ asdefined above, with the proviso that when R₁ is a lower alkyl orarylalkyl, Z′ is hydrogen or C₁-C₄ alkoxy and T₁, T₂, T₃, and T₄ are allC, Y cannot be hydrogen or alkyl; and

(ii) carbamyl, cyano, vinyl, nitro, sulfamyl, or sulfamido; and

R₃ is selected from the group consisting of hydrogen, hydroxyl, methyl,C₁-C₃ hydrocarbon chain or C₁-C₃ alkoxy, allyl and amino;

alternatively R₁ together with Z′, Y′ together with R₃, R₃ together withY, or Z′ together with Y′ can form an aromatic or aliphatic ringstructure, optionally heterocyclic, optionally substituted with 1-4R^(11,)

wherein the target has an active site and catalytic mechanism similar toapopain and rhinovirus 3C protease.

Preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen, hydroxyl, methoxy, or —OCH₂CONH₂; Y is hydrogen,halogen nitro, cyano, methyl, or —COOCH₂CH₃ with the proviso that whenZ′ is hydrogen or methoxy, Y cannot be hydrogen; Y′ is hydrogen,halogen, hydroxyl, methyl or methoxy; and R₃ is hydrogen, hydroxyl,methyl or methoxy. Exemplary compositions having this orthohydroxy ketoaryl core structure include compositions wherein: (1) Z′ and R₃ arehydrogen and Y and Y′ are Cl or Br; (2) Z′ and R₃ are hydrogen, Y isnitro, and Y′ is Cl or methyl; (3) Z′, Y′ and R₃ are hydrogen and Y iscyano; (4) Z′ and R₃ are hydroxyl, Y is hydrogen or —COOCH₂CH₃, and Y′is hydrogen; (5) Z′ is hydroxyl, R₃ is hydrogen, and Y and Y′ are Cl;and (6) Z′ is —OCH₂CONH₂, and R₃, and Y′ are hydrogen.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen, hydroxyl, C₁-C₄ alkoxy or —OCH₂CONH₂; Y ishydrogen, halogen, hydroxyl, nitro, cyano, methyl or —COOCH₂CH₃ with theproviso that when Z′ is hydrogen or C₁-C₄ alkoxy, Y cannot be hydrogenor methyl; Y′ is hydrogen, halogen, hydroxyl, nitro, methyl or methoxy;and R₃ is hydrogen, hydroxyl, methyl or C₁-C₃ alkoxy.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen, hydroxyl, methoxy or —OCH₂CONH₂; Y′ is hydrogenor halogen; R₃ is hydroxyl or methoxy; and Y is hydrogen, halogen orhydroxyl with the proviso that when Z′ is hydrogen, Y cannot behydrogen. Exemplary compositions having this orthohydroxy keto aryl corestructure include compositions wherein: (1) Z′ is hydroxyl, R₃ ismethoxy, and Y′ and Y are hydrogen or Cl; (2) Z′ is methoxy, R₃ ishydroxyl, Y′ is hydrogen or Cl, and Y is hydroxyl or Cl; (3) Z′ ishydrogen, R₃ is hydroxyl, and Y′ is hydrogen or Cl and Y is Cl; and (4)Z′ is —OCH₂CONH₂, R₃ is methoxy, and Y′ and Y are Cl.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen or hydroxyl; Y′ is hydrogen, nitro, Cl or I; R₃is hydrogen or hydroxyl; and Y is hydrogen, Cl or I. Exemplarycompositions having this orthohydroxy keto aryl core structure includecompositions wherein: (1) Z′ and R₃ are hydroxyl, and Y′ and Y arehydrogen; (2) Z′ and R₃ are hydrogen, and Y′ and Y are Cl or I; and (3)Z′, R₃ and Y are hydrogen and Y′ is nitro.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ and R₃ are hydrogen; and W is CH₂-phenyl, CH₂CH₂-phenyl or(CH₂)₇CH₃.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein R₃ and Y are hydrogen.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′, Y′ and Y are hydrogen and R₃ is methoxy.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen; Y is hydrogen; Y′ is hydrogen, methyl, orhalogen; and R₃ is hydrogen or methyl. Exemplary compositions havingthis orthohydroxy keto aryl core structure include compositions wherein:(1) Z′, R₃ and Y are hydrogen and Y′ is hydrogen, methyl, Cl or Br; and(2) Z′ and Y are hydrogen, R₃ is methyl, and Y′ is Cl.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Y′, R₃, and Y are hydrogen.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is OH, Y′ and Y are H, and R₃ is methyl.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Z′ is hydrogen or hydroxyl; Y is halogen; Y′ is hydrogen orhalogen; R3 is hydrogen or hydroxyl; and R¹¹ is hydrogen or hydroxyl.Exemplary compositions having this orthohydroxy keto aryl core structureinclude compositions wherein: (1) Z′ is hydrogen, R₃, and R¹¹ arehydrogen or hydroxyl, Y′ is hydrogen or Cl, and Y is Cl; (2) Z′ ishydroxyl, R₃ and R¹¹ are hydrogen, and Y′ and Y are hydrogen or Cl; and(3) Z′, R₃ and R¹¹ are hydroxyl, and Y′ and Y are hydrogen.

Other preferred chemical compositions for these methods of the presentinvention have the following orthohydroxy keto aryl core structure

wherein Y is hydrogen or Cl; Y′ is hydrogen or Cl; R₃ is hydrogen orhydroxyl; R₁₁ is hydrogen or hydroxyl; and R₁₂ is hydrogen or hydroxyl.Exemplary compositions having this orthohydroxy keto aryl core structureinclude compositions wherein: (1) R₃, R¹¹ and R¹² are hydrogen orhydroxyl, and Y′ and Y are hydrogen or Cl; (2) R₃ is hydroxyl, Y′ and Yare hydrogen, and R¹¹ and R¹² are hydrogen or hydroxyl; and (3) R₃ ishydroxyl, Y′ and Y are Cl, and R¹¹ and R¹² are hydrogen.

In another aspect, the invention is a composition having the followingstructure

wherein R₁ is hydrogen, a hydrocarbon chain from 1 to about 10 carbonsoptionally substituted with an aryl, C₁-C₃ alkoxy, amino optionallysubstituted with C₁-C₁₀ hydrocarbon or C₁-C₃ arylalkyl, aryl optionallysubstituted with hydroxyl or keto, or arylalkyl optionally substitutedwith hydroxyl; R₃ is hydrogen, hydroxyl, methyl, or C₁-C₃ alkoxy; Y′ ishydrogen, hydroxyl, halogen, nitro, cyano, C₁-C₃ alkyl, or C₁-C₃ alkoxy;and Y is hydrogen, hydroxyl, halogen, nitro, cyano, or COOCH₂CH₃.Exemplary compositions having this orthohydroxy keto aryl core structureinclude compositions wherein: (1) R₁ is methyl, and Y′, R₃ and Y arehydrogen, thereby comprising 2-(2-acetyl-3-hydroxyphenoxy)-acetamide;and (2) R₁ is (CH₂)₄CH₃, R₃ is methoxy, and Y and Y′ are Cl.

The term “amino acid” used above is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including for example hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids. The term“oligopeptide” refers to a series of amino acids linked by peptidebonds.

Suitable sequences of amino acids can be chosen according to theteachings of Cordingley et al., J. Biol Chem., 265(16):9062-9066 (1990).Additionally, various amino acids may be screened as follows: (1) adesirable protease, such as the 3C picornavirus protease is immobilizedto a solid support, (2) candidate sequences are brought into contactwith the immobilized protease, (3) residues that bind to theimnmobilized proteases are chosen as candidate sequences.

The pharmaceutical compositions of the present invention are suitablefor the treatment of diseases manifested by the activity of cysteineproteases of the CB Clan (families C3, C4, C24, C30, C37 and C38) of theCD Clan (family C14), of the CE clan and of family C13.

The term “diseases, which manifestation is dependent on the activitycysteine proteases”, refers to a disease that can be treated, prevented,alleviated or cured by inhibition of cysteine proteases of the CB Clan,the CD Clan, the CE clan and the C13 family. Preferably, the inhibitionis of “picornavirus 3C-like cysteine proteases”, which are cysteineproteases having an active site similar to the active site of the 3Cprotease, (a catalytic dyad of Histidine and Cysteine) and mostpreferably of 3C-cysteine proteases.

Thus, most preferably, the pharmaceutical compositions of the inventionaccording to the inhibition aspect of the invention are for thetreatment of viral infections and of diseases wherein excessiveapoptosis is implicated and wherein apoptosis should be reduced mostpreferably for picornaviral infection, neurodegenerative disease andcertain cardiovascular diseases.

The pharmaceutical compositions of the invention according to theinhibition aspect of the invention are suitable for the treatment ofcommon colds, allergic rhinitis, poliomyelitis, hepatitis-A,encephalitis, meningitis, hand-foot-and-mouth disease,encephalomyocarditis, summer flu (enteroviral upper respiratoryinfection), asthma, various allergies, myocarditis, acute hemorrhagicconjunctivitis, disseminated neonatal infection and Borhnolm's disease.All the above are diseases which manifestation is dependent on theactivity of a cysteine protease of the CB clan.

The inhibitors of the pharmaceutical compositions of the presentinvention selectively bind to the picornaviral proteases, essentially ina similar manner as the viral coded natural substrate of the proteases,and compete with the substrates for proteases. This competition servesto inhibit viral maturation and thus to inhibit disease progression invivo.

The pharmaceutical compositions of the present invention are suitablealso for the treatment of diseases manifested by the activity of thecysteine proteases of the CD clan, i.e apoptosis-involved diseases,which includes activation, as in cancer, as well as inhibition (as inneurodegenerative diseases) of apoptosis.

The pharmaceutical compositions of the present invention are alsosuitable for the treatment of adenovirus-involved diseases.

Thus the present invention, according to its inhibition aspect, furtherprovides a method for treatment of viral infection, in particular apicornaviral infection by administrating to a subject in need of suchtreatment a pharmaceutically acceptable amount of a compound of formulae(I) to (VI), which has protease inhibitor activity optionally togetherwith a pharmaceutically acceptable carrier.

The present invention further concerns, according to its inhibitionaspect, a method for the treatment of cardiovascular diseases such asischemic cardiac damage by administering to a subject, in need of suchtreatment, a pharmaceutically acceptable amount of a compound offormulae (I) to (VI), which has protease inhibitor activity optionallytogether with a pharmaceutically acceptable carrier.

Further, the present invention, according to its inhibition aspect,provides a method for the treatment of neurodegenerative diseases suchas Alzheimer's, Parkinson's and Huntington's, a pharmaceuticallyacceptable amount of a compound of the formulae (I) to (VI), which hasprotease inhibitor activity optionally together with a pharmaceuticallyacceptable carrier.

As will no doubt be appreciated by the person skilled in the art, theabove Formulae I-VI cover a large number of possible compounds, some ofwhich are inhibitors and some are enhancers, and from those which areinhibitors some are more effective inhibitors of cysteine proteases ofthe above types than others

In order to determine which of the compounds are suitable as 3C proteaseinhibitors, according to the inhibitory aspect of the present invention,compounds may be screened for inhibitory activities according to one ofthe following assays:

Assays for screening picornaviral protease inhibitors

I. Birch et al. (Protein Expression and Purification, 6:609-618 (1995))have developed a continuous fluorescence assay to determine kineticparameters and to screen potential HRV14 3C protease inhibitors. Theassay consists of a consensus peptide for rhinoviruses connected to afluorescence donor group (anthranilic acid, Anc) at the N terminal andto an acceptor group (p-NO₂-Phe; Pnp) at the P4 position, both groupsflanking the scissile bond (Gln/Gly). The substrate peptide consists ofthe following sequence: Anc-Thr-Leu-Phe-Gln-Gly-Pro-Val-Pnp-Lys. Thereis a linear time dependent increase in fluorescence intensity as thesubstrate is cleaved, which allows continuous monitoring of thereaction. Multiwell plates containing one inhibitor per well allows forrapid screening by measuring the fluorescence intensity in each well.

II. Heinz et al. (Antimicrobial Agents and Chemotherapy, 267-270 (1996))developed an assay method for measuring 3C protease activity andinhibition using the substratebiotin-Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu-Lys-fluorescein-isothiocyanate.Cleavage mixtures containing inhibitors are allowed to bind to avidinbeads and are subsequently washed. The resultant fluorescence of thebead is proportional to the degree of inhibition.

III. Another assay developed by McCall et al. (Bio/Technology, 121012-1016 (1994)) measures in addition to the inhibitory effects of thecandidate inhibitors, their capability to enter into cells so that ahigh capacity screen for compounds inhibiting the 3C protease of HRV-1Bis developed. The assay uses a recombinant strain of E-coli expressingboth the protease and a tetracycline resistance gene modified to containthe minimal 3C protease cleavage sequence. Cultures growing inmicrotiter plates containing tetracycline are treated with potentialinhibitors. Culture with no inhibition of the 3C protease, show reducedgrowth due to cleavage of the essential gene product. Normal growth isseen only in cultures that contains an effective 3C protease inhibitor.

IV. An assay was developed in our lab based on a protein consisting ofthe 3C protease fused to DHFR. The cleavage of the fusion protein byexternal 3C protease (type 1A) is monitored by gel-electrophoresis. Thedegree of cleavage is proportional to the ratio of low molecular weightproteins (3C and DHFR) to intact fusion protein, as observed on the gel.

V. Other assays developed for inhibition of other cysteine proteases arewell known in the art.

The pharmaceutical compositions of the invention according to the“enhancement aspect” are suitable for diseases manifested by deficientapoptosis, i.e. inappropriate activity of Apopain and of other “3Cprotease-like proteins”. The term “3C protease-like proteins” refers tocysteine proteases with active site structures similar to that of apicornavirus 3C protease as discovered by homology or by x-ray analysis.An example of such a protease is Apopain. In particular, the diseasesare characterized by insufficient apoptosis and include among othersautoimmune diseases, viral-caused infectious diseases and certain typesof cancer.

The diseases may be treated, prevented, alleviated or cured bycompositions of the invention having Apopain enhancing activity.

Diseases in which insufficient apoptosis is implicated will be cured byApopain enhancement leading to normal or excessive levels ofapoptosis—thus, for example, certain cancers originating from subnormallevels of programmed cell death will be eliminated following therestoration of normal levels of apoptosis or the establishment of higherthan normal levels of apoptosis.

The present invention further concerns a method for treatment ofautoimmune diseases, viral-caused infectious diseases and certain typesof cancer as well as cardiovascular diseases such as ischemic cardiacdamage by administering to a subject in need of such treatment apharmaceutically acceptable array of a compound of formulae I-VI, whichhas protease enhancing activity

As will be no doubt appreciated by a person skilled in the art, formulaeI-VI above cover a large number of possible compounds, some of which areinhibitors and some are enhancers, and from those which are enhancerssome are more effective than others.

In order to determine compounds that are most suitable as enhancers,compounds may be screened by the following additional assay (VI)

VI: Apopain (Caspase-3) Regulation Assay

The FluorAce™ Apopain Assay Kit (Bio-rad) was employed in multiwellformat. Compounds assayed were diluted 5-fold in distilled water (fromstock solutions in ethanol or DMSO) and centrifuged (5′, 14K rpm). 10 μLof the supernatant was further diluted in wells containing 100 μLdistilled water and 40 μL 6×Buffer (41.7 mM PIPES, pH 7.4, 8.3 mM EDTA,0.42% CHAPS, 20.8 mM DTT). This process was carried out twice to yieldduplicate wells for each compound. Control wells were prepared byaddition of EtOH and DMSO (5-fold diluted in distilled water) into wellscontaining 6×Buffer and distilled water (40 μL and 100 μL,respectively), to form 8 wells with each solvent. Enzyme (stock solution10-fold diluted in distilled water, see Bio-rad booklet 4100119) wasadded to one set of compound-containing cells and to 8 control wells (4with ethanol and 4 with DMSO). The plate was preincubated for ˜80′ at27°-28° C. Substrate (Z-DEVD-AFC, 490 μM, 40 μL) was then added to allwells. The plate was left at room temperature and fluorescence(360/40↑530/20↓) was measured at several time points (FL500 fluorimetricreader, Bio-tek instruments). Fluorescence at enzyme-containing wellswas background-subtracted at each time point and initial rates weredetermined by linear regression (typically R²>0.97). Percentageinhibition and activation was determined by relating the slope withcompound to the control slope (with appropriate solvent). IC50 valuesare extrapolated.

Pharmaceutically acceptable carriers are well known in the art and aredisclosed, for instance, in Sprowl's American Pharmacy, Dittert, L.(ed.), J. B. Lippincott Co., Philadelphia, 1974, and Remington'sPharmaceutical Sciences, Gennaro, A. (ed.), Mack Publishing Co., Easton,Pa., 1985.

Pharmaceutical compositions of the compounds of the present invention,or of pharmaceutically acceptable salts thereof, may be formulated assolutions or lyophilized powders for parenteral administration. Powdersmay be reconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution, but a lipophiliccarrier, such as propylene glycol optionally with an alcohol, can bemore appropriate for compounds of this invention. Examples of suitablediluents are normal isotonic saline solution, standard 5% dextrose inwater of buffered sodium or ammonium acetate solution. Such aformulation is especially suitable for parenteral administration, butcan also be used for oral administration or contained in a metered doseinhaler of nebulizer for insufflation. It may be desirable to addexcipients such as ethanol, polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride orsodium citrate

Alternately, the compounds of the invention may be encapsulated,tableted or prepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, soy bean oil, peanutoil, olive oil, glycerin, saline, ethanol, and water. Solubizing agents,such as dimethylsulfoxide, ethanol or formamide, may also be added.Carriers, such as oils, optionally with solubizing excipients, areespecially suitable. Oils include any natural or synthetic non-ionicwater-immiscible liquid, or low melting solid capable of dissolvinglipophilic compounds. Natural oils, such as triglycerides arerepresentative. In fact, another aspect of this invention is apharmaceutical composition comprising a compound of formula (I) and anoil.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Solubilizing agents, such as dimethylsulfoxide or formamide,may also be added. The carrier may also include a sustained releasematerial such as glyceryl monostearate or glyceryl distearate, alone orwith a wax. The pharmaceutical preparations are made following theconventional techniques of pharmacy involving milling, mixing,granulating, and compressing for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation can beadministered directly p.o. or filled into a soft gelatin capsule.

For rectal administration, a pulverized powder of the compounds of thisinvention may be combined with excipients such as cocoa butter,glycerin, gelatin or polyethylene glycols and molded into a suppository.The pulverized posers may also be compounded with an oily preparation,gel, cream or emulsion, buffered or unbuffered, and administered througha transdermal patch.

Nasal administration of the compounds of the invention can also be usedespecially for the treatment of common cold and allergic rhinivity.

The present invention also concerns a method for the detection ofpicornaviral infection. According to a method of the invention, acompound of the invention bearing a detectable label (for exampleattached to one of its substituents) is incubated with a samplesuspected of containing picornaviruses, under conditions enablingbinding of the compound to proteases. Preferably, the sample should betreated with a lysing agent in order to release the picornavirusproteins from inclusion bodies. Then it is determined whether thelabeled compounds of the invention are bound to any proteins in assay. Apositive answer (beyond a predetermined control level) is indicative ofthe presence of a picornavirus in the assayed sample.

The present invention further concerns several novel chemical compoundsdenoted in the examples as follows:

SA#121, SA#132, SA#116, SA#118, SA#134, SA#135, SA#120, SA#127, SA#128,SA#15*, SA#16*, SA#107*, SA#108*, SA#110**, SA#43, SA#109*, SA#139,SA#51.

previously described

commercially available

The invention will now be described in reference to some non-limitingexamples.

DETAILED DESCRIPTION OF THE INVENTION

Syntheses

The numbers in the following examples represent preferred methods forsynthesizing the compounds discussed herein. Thus, the compounds claimedwithin should not be construed to be limited by its respective method ofsynthesis. Additionally, each example refers to the number of thecompound as appears in the tables preceded by SA#.

Example 116

202 mg (0.97 mmol) of 2,4-dihydroxyhexanophenone (Aldrich, 96%) wascompletely dissolved in methylene chloride (anhydrous, 8 mL), containing2 equivalents of methanol (81 μL, 1.98 mmol, added by pipette). 2 mL ofa freshly prepared 1M solution of sulfuryl chloride in anhydrousdichloromethane was added to the light brown solution at roomtemperature (24° C.) while stirring. The color of the solution turnedslightly yellow immediately, and the yellowness deepened 15 minuteslater. At this stage the starting material was completely consumed, asshown by reverse-phase HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm).The reaction was continued for 45 minutes further, after which thesolvents were flash-evaporated to give beige powder (269 mg; ˜100%yield), which was dried under high vacuum overnight. An analyticalsample was purified by crystallization from CDCl₃ (−10 mg/mL with activecharcoal, white needles). M.P. (Corr.): 102°-103° C. IR (KBr disk)ν1629.7 cm⁻¹ (strong). HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm):R_(t)=1.9′ (100%). ¹H NMR (CDCl₃): δ13.31 (s, 1H), 7.69 (s, 3H),7.04-5.44 (broad s, 1H), 2.88 (t, 2H, J=7 Hz), 1.84-1.54 (m, 2H, J=7Hz), 1.54-1.14 (m, 4H), 0.88 (t, 3H). ¹³C NMR (CDCl₃, 60 MHz, adjacenthydrogens by parallel DEPT): 205.6 (q), 159.4 (q), 154.5 (q), 129.3 (t),114.4 (q), 111.9 (q), 109.6 (q), 38.6 (s), 31.7 (s), 24.6 (s), 23.0 (s),14.5 (p). MS (EI⁺): 276.0 (M⁺, 19%), 205.0 (M⁺—C₅H₁₁, 2 chlorine atoms,100%).

Example 120

A solution of 3,5-dinitrosalicylic acid (1140 mg, 5.0 mmol) indichloromethane (˜10 mL) was treated with excess PCl₅ at roomtemperature for 40 minutes. The methylene chloride was removed by rotaryevaporation and the resultant oil was washed with hexanes (3 times),dissolved in methylene chloride (10 mL) and cooled to 0° C. 1.1Equivalents of triethylamine (780 μL, 5.5 mmol) and benzylamine (600 μL,5.5 mmol) were introduced by pipette and the reaction mixture wasgradually warmed to room temperature in the course of 40 minutes. Thesolution was neutralized by addition of 5% aqueous 11Cl and extractedtwice with ethyl acetate. The organic phases were combined andevaporated. The residue was washed with petrol ether and cooled,resulting in a viscous brown oil. The crude oil was purified by flashchromatography (0-10% EtAc petrol ether 60-80) Fraction 5 (4% EtAc)contained pure material (TLC, reverse phase HPLC) and was evaporated andleft overnight in-vacuo (on an oil pump). The orange oil obtained wastaken up in methylene chloride and washed with aqueous K₂CO₃. A fineyellow-orange sediment appeared (540 mg, ˜30% yield), which wasdetermined to be pure by reverse phase HPLC (30% acetonitrile in H₂O, 1mL/min, 256 nm, R₁=2.2′). ¹H NMR (CD₃OD, 200 MHz): δ8.99 (d, 1H, J=3.2Hz), 8.82 (d, 1H, J=3.2 Hz) 7.35-7.05 (m, 5H), 3.64 (t, 2H, J=7.4 Hz),2.92 (t, 2H, J=7.4 Hz). MS (FAB⁺):318.1 (MH⁺, 76%).

Example 121

Williamson ether synthesis was carried out in accordance with theprocedure of Burgstahler & Worden (Organic Synthesis, Coll. Vol. V,1973, pp. 251-4).

2,6-dihydroxyacetophenone (Sigma, 1.52 g. 10.0 mmol), chloroacetamide(Aldrich, 953 mg, 10.2 mmol) and distilled water (5.5 mL) were added toa 25 mL round-bottomed flask equipped with a magnetic stirrer and awater-jacketed reflux condenser. The turbid suspension was heatedrapidly on an oil bath set at 110° C. and 2 mL of an aqueous 5N NaOHsolution was added (pipette). A clear dark orange solution formed withinthe flask immediately. The reaction mixture was left overnight underlight reflux and cooled with tap water (oil-bath temperature between110° C. and 114° C.). The dark red solution was cooled to roomtemperature and a red oil separated beneath an orange solution. 5N NaOHwas added (1 mL) and the solution was decanted. The oil was dried underhigh vacuum, dissolved in ethyl acetate. 438 mg were purified by columnchromatography (SiO₂, ethyl acetate, 197×21 mm, 24 g, 1.5 drops/minute).Fractions 10-12 (bright yellow-orange) were pooled and yielded anevaporation orange solid (106 mg). M.P. (Corr.): 151°-152° C. HPLC (70%acetonitrile in H₂O, 1 mL/min. 256 nm): R_(t)=2.4′ (100%). ¹H NMR(DMSO-d⁶): δ11.78 (broad s, 1H), 7.44 (broad s, 2H), 7.30 (t, 1H, J=8.3Hz), 6.53 (d, 1H, J=8.3 Hz), 6.44 (d, 1H, J=8.3 Hz), 4.53 (s, 2H), 2.60(s, 3H). MS (EI⁺): 209.1 (M⁺, 55%), 137.0 (M⁺—(CH₂)₂CONH₂, 100%).

Example 127

A solution of 3,5-dinitrosalicylic acid (839 mg, 3.68 mmol) indichloromethane (10 mL) was treated with excess PC₅ at room temperaturefor 30 minutes. The mixture was filtered through cotton wool, themethylene chloride evaporated and the resultant oil washed 3 times withhexanes, dissolved in methylene chloride (10 mL) and cooled to 0° C. 1.1Equivalents of triethylamine (575 μL, 4.05 mmol) and of n-octylamine(510 μL, 4.06 mmol) were introduced by pipette and the reaction mixturewas brought to room temperature in the course of 30 minutes. Thesolution was neutralized by the addition of 5% aqueous HCl and themethylene chloride layer was separated, dried with MgSO₄ and filteredthrough cotton wool. The clear filtrate was mixed with silica gel 60H,evaporated, loaded on a column and separated by flash chromatographywith a petrol ether (b.p. 60-80) forerun (200 mL) followed by a smoothgradient of 5-20% EtAc in petrol ether.

Fractions containing product were pooled, dried by flash-evaporation andwashed with aqueous sodium carbonate. The solvent was decanted and thesedimented orange particles were filtered with a glass sinter, washedwith ice-cold water, powdered with a glass rod, washed with methylenechloride and grounded once more yielding 360 mg of bright orange powder(30% yield), pure by HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm,R_(t)=1.28′). ¹H NMR (CD₃OD, 200 MHz): δ8.99 (d, 1H, J=3.2 Hz), 8.82 (d,1H, J=3.2 Hz) 7.35-7.05 (m, 5H), 3.64 (t, 2H, J=7.4 Hz), 2.92 (t, 2H,J=7.4 Hz). MS (FAB⁺): 332.1 (MH¹, 100%),

Example 128

A solution of 3,5-dinitrosalicylic acid (1040 mg, 4.56 mmol) indichloromethane (15 mL) was treated with excess PCl₅ at room temperaturefor 45 minutes. The mixture was filtered through cotton wool and thesolvent was evaporated. The resultant viscous red oil cooled to 0° C. onan ice-water bath and washed twice with scrubbing with hexanes. Thewashed acid chloride was resuspended in methylene chloride at 0° C., and1.1 Equivalents of triethylamine (830 μL, 5.00 mmol) and of n-octylamine(712 μL, 5.03 mmol) were introduced by pipette. The reaction mixture wasbrought to room temperature in the course of 4 hours, at the end ofwhich 20mL 5% aqueous HCl was added. The methylene chloride phase wasseparated, mixed with silica gel 60H, evaporated, loaded on a column andseparated by flash chromatography with 2% EtAc in petrol ether 60-80.Fractions containing product were pooled, dried by flash-evaporation andwashed with aqueous sodium carbonate. The sediment was filtered in-vacuoand washed with methylene chloride. The orange sediment thus obtainedwas purified by reverse phase HPLC (30% acetonitrile in H₂O, 1 mL/min,256 nm, R_(t)=1.37′). ¹H NMR (CD₃OD, 200 MHz): δ9.02 (d, 1H, J=4 Hz),8.80 (d, 1H, J=4 Hz) 3.46 (t, 2H, J=8 Hz), 1.80-1.60 (m, 2H), 1.60-1.12(m, 10H), 0.91 (t, 3H, J=8 Hz). MS (FAB⁻): 340.2 (MH⁻, 100%), 362.2(MNa⁺, 24%), 378.1 (MK⁺, 32%).

Example 132

2,4,6-trihydroxybenzoic acid (2.00 g, 10 mmol) and CaCl₂-distilledethanol (15.5 mL) were added to a three-necked flask equipped with areflux condenser, a calcium chloride trap and a rubber septum.Boron-trifluoride-etherate (1.5 mL) was added by syringe to thebright-colored solution, and scant white fumes were observed. Thesolution was refluxed overnight with stirring at room temperature. Theyellow-orange solution was decomposed by addition of water (50 mL) andwas extracted with ether (3×50 mL) and ethyl acetate (2×50 mL).Evaporation of the ethyl acetate extract yielded an orange pasty solidcontaining the desired ethyl trihydroxybenzoate contaminated withphloroglucinol, and other minor side-products (1.2 g). Isolation of theester was achieved by column chromatography (silica gel 60; 15%-50%ethyl acetate/petroleum ether 60-80). Fractions 12-13 (50% ethylacetate) were pooled and evaporated to yield 341 mg of 85% pure(reverse-phase HPLC, 70% acetonitrile in H₂O, 1 mL/min, 256 nm,R_(t)=2.25′) ethyl trihydroxybenzoate (NMR). This material was driedin-vacuo and subjected to a hoesch condensation (procedure by Whalley,J.Chem Soc., 1951, 3229). 340 mg (˜1.5 mmol) were placed in aflame-dried air-jacketed 2-necked flask equipped with an in-situ HClgas-generating system (H₂SO₄ equal pressure funnel. Kipp apparatus withNH₄Cl, connected in tandem to H₂SO₄ and air traps) was dissolved in 50mL sodium dried ether. Oven-dried ZnCl₂ (0-8 g) and AlCl₃, (anhydrous,under argon) were added to the clear solution. Upon addition of AlCl₃ avigorous reaction ensued and the solution turned immediately turbidyellow. Acetonitrile (HPLC grade; 2 mL, 38 mmol) was added and a drystream of dry hydrogen chloride gas was passed through the mixture. Thesolution became clear within a few minutes, was turbid again after onehour and was left overnight at room temperature. The ethereal solutionwas filtered in-vacuo and the white solid (ketimine hydrochloride) wasdissolved in water (25 mL) and hydrolysed by heating on a hot plate. Theaqueous solution was concentrated to 5 mL and cooled. Upon cooling,needles appeared which were kept overnight at 4° C. Cold filtrationyielded sweet-odored yellow needles, which were dried intensively in aflash evaporator (12 mg). The material was 72% pure by reverse-phaseHPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm, R_(t)=3.50′). ¹H NMR(CD₃OD, 200 MHz): δ5.97 (s, 1H), 4.13 (q, 2H, J=7.0 Hz), 2.63 (s, 3 H),1.51 (t, 3 H, J=7.0 Hz).

Examples 134, 135

14 mg of 2,4-dihydroxy-6-methoxy-hexanophenone (Example 108 in PCT, 0.06mmol) was dissolved in methylene chloride (anhydrous, 1 mL) containing20 μL of methanol. 120 μL of a 1M solution of sulfuryl chloride inanhydrous dichloromethane was added to the stirred opaque solution via ahamilton syringe. A faint yellow color appeared and the solution wasleft in open air with stirring overnight. Yellow needles resided on thesides of the flask (16 mg, ˜100% yield) and were purified by columnchromatography (SiO₂, 20% ethyl acetate in petrol ether 60-80, 50×5 mm,1 g, 0.5 mL fractions). Evaporation of fraction 5 yielded approximately˜2 mg yellow flakes corresponding to example 134. HPLC (70% acetonitrilein H₂O, 1 mL/min, 256 nm, R_(t)=5.45′). ¹H NMR (CDCl₃, 200 MHz): δ13.23(s, 1H), 6.43 (s, 1H), 6.2-6.0 (broad s, 1H), 3.91 (s, 3H), 3.06 (t, 2H,J=7.4 Hz), 1.8-1.6 (m, 2H), 1.4-1.2 (m 4H), 0.91 (t, 3 H, J=6.4 Hz). MS(EI⁺): 272.1 (MH⁻, 15.5%), 201.0 (M⁺—C₅H₁₁, 1 chlorine atom, 100%).

Fraction 7 yielded upon evaporation ˜3 mg of pure example 135 asyellowish flakes. HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 mn,R_(t)=5.78′). ¹H NMR (CDCl₃, 200 MHz): δ14.63 (s, 1H), 6.14 (s, 1H),3.88 (s, 3H) 3.00 (t, 2H, J=7.4 Hz), 1.8-1.5 (m, 2H), 1.5-1.3 (m, 4H),0.91 (t, 3H).

Example 139

Synthesis was carried out essentially as described above in example 121.With the main difference being the temperature at which the base isadded (room temperature vs. 100° C.). 131 mg (0.86 mmol) of2,5-dihydroxyacetophenone (Sigma) was placed in a 5 mL glass trapequipped with a magnetic stirrer and connected to a water-cooled refluxcondenser 82 mg chloroacetamide (0.88 mmol), 0.17 mL NaOH (5N) and 1.2mL H₂O were added to the reaction vessel. The orange solution was heatedrapidly on an oil bath (oil temperature of 106°-110° C.) and mild refluxensued. The reaction was left overnight (24 h) in reflux and cooled toroom temperature resulting in a dark brown solution with sediments onthe vessel walls. The pH was found to be neutral, and the solution wasdried in high vacuum. Pure material (HPLC, TLC) was obtained by columnchromatography (SiO₂, 40%-100% ethyl acetate in petrol ether 60-80,128×12 mm, 5 g). Fractions 12-15 (EtAc) were pooled and evaporated toyield 7 mg of a yellow solid. HPLC (70% acetonitrile in H₂O, 1 mL/min,256 nm, R_(t)=2.5′, 96%). ¹H NMR (DMSO-d⁶, 200 MHz): δ12.2-10.8 (broads, 1H), 7.54 (broad s, 1H), 7.39 (d, 1H, J=3.2 Hz), 7.22 (dd, 1H, J₁=9.5Hz, J₂=3.2 Hz), 6.92 (d, 1H, J=9.5 Hz), 4.42 (s, 2H), 2.62 (s, 3H).

Example 43

256 mg (1.13 mmol) of 2′-hydroxy-3-phenylpropiophenone (Aldrich) weredissolved in methylene chloride (8 mL) containing 2 equivalents ofmethanol (91 μL) in a 25 mL round bottom flask equipped with a magneticstirrer and an 8″ reflux condenser. 2.26 mL sulfaryl chloride (1M/methylene chloride, freshly prepared) was added to the clear solution(by glass pipette), and the color intensified. The mixture was stirredat room temperature (21° C.) for 4 hours. A major product was thenobserved by reverse-phase HPLC (70% acetonitrile in H₂O, 1 mL/min, 256nm), amounting to 55% of the total mixture. An additional amount ofsulfuryl chloride 1M solution was added (0.56 mL, 0.5 eq.) and themixture was stirred at room temperature for 25 hours after which HPLCshowed an increase in major product (72%). Solvents were evaporated(water bath at 67° C.), resulting in a yellow oil (384 mg, ˜100% yield).Upon cooling, a milky solid formed with orange droplets. The crudemixture was dissolved in 2% ethyl acetate/petrol ether 40-60, with a fewdrops of methylene chloride. The pale yellow solution (with few orangedroplets) was purified by column chromatography (−8 g SiO₂, 2% ethylacetate in petrol ether 40-60, 185×12 mm, −3 mL fractions). Fractions 6and 7 were evaporated to yield 22 mg of purified product (HPLC), whichis a colorless oil having a strong scent.

HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm): R_(t)=12.9′. ¹H NMR(DMSO-d⁶, trace CDCl₃, 200 MHz): δ11.63 (s, 1H, sharp), 7.85 (d, 1H,J=2.3 Hz), 7.52 (dd, 1H, J₁=8.5 Hz, J₂=2.3 Hz), 7.38-7.05 (m, 5H), 7.00(d, 1H, J=8.7 Hz), 3.43 (t, 2H, J=7.4 Hz), 2.92 (t, 2H, J=7.5 Hz). MS(EI⁺): 260.1 (M⁺, 62%, 1 chlorine atom), 155.1 (M⁻—(CH₂)₂Ph, 100%, 1chlorine atom).

Example 109

Absolute methanol (325 μl. 2 eq.) and dichloromethane (8 ml) were addedto 2,6-dihydroxyacetophenone (152 mg, 1 mmol) in a 25 mL round bottomflask equipped with a magnetic stirrer to provide an almost clear orangesolution. Sulfuryl chloride was added at room temperature (0.65 mL) anda yellow solid appeared. Dichloromethane was added (10 mL) to form asolid suspension. Filtration in-vacuo provided pale yellow and brownairy chunks (622 mg). The filtrate provided an additional amount (139mg) forming a total yield of about 87%. Recrystallization of the majorfraction from ethanol provided clumps of small yellow needles (187 mg).Recrystallization of the filtrate from EtOH-water provided off-yellowishsheaths (91 mg). HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm):R_(t)=4.6′. ¹H NMR (CDCl₃, 200 MHz): δ10.05 (sharp s, 1H), 7.54 (s, 1H),2.79 (s, 4H). MS (EI⁺): 220.0 (M⁺, 44%), 205.0 (M⁺—CH₃, 97%).

Example 15

A stirred solution of 2,6-dihydroxy-4-methoxyhexanophenone (cf. example107, 28 mg, 0.12 mmol) in absolute methanol (0.25 ml), was diluted inanhydrous dichloromethane (10 ml) at room temperature. A 1M solution ofsulfuryl chloride in anhydrous dichloromethane was added dropwise (0.26ml, 2.2 eq.). Within a few seconds, the color of the resultant mixturewas observed to change from faint yellow to bright vivid yellow. 20minutes later, the solvents were evaporated and residual sulfurylchloride was removed in vacuo, resulting in yellow crystals (34 mg,quantitative). M.P. 101° C. ¹H NMR (CDCl₃, 200 MHz): δ10.32 (s, 2H),3.98 (s, 3H), 3.13 (t, 2H), 1.71 (t, 2H), 1.35 (m, 4H), 0.91 (t, 3H)[identical to spectrum described by Masento et al., 1998, Biochem. J.256: 23-28], MS (EI⁺): 306.0 (M⁺, 21% ), 235.0 (M⁺—C₅H₁₁, 100%).

Example 16

2,4-dihydroxy-6-methoxyhexanophenone (cf. example 108; 10 mg, 0.04 mmol)was reacted with sulfuryl chloride in a manner analogous to thatdescribed in example 15 above. The product was vaccuum-dried resultingin orange crystals (12 mg). ¹H NMR (CDCl₃, 200 MHz): δ14.02 (s, 1H),6.51 (s, 1H), 3.91 (s, 3H), 3.05 (t, 2H), 1.69 (t, 2H), 1.32 (m, 4H),0.89 (t, 3H).

Examples 107 and 108

Dihydroxyanisole (1.113 g, 7.9 mmol), hexanonitrile (1.60 ml, 13.2 mmol)and zinc chloride (700 mg) were dissolved in 50 ml of sodium-driedether. The stirred solution was saturated with a steady stream of dryhydrogen chloride gas and after 5 minutes turned milky. 10 minuteslater, a viscous orange oil separated and the mixture was leftovernight. The colorless solution was decanted and the oil was taken in50 ml of ice-cold water. The resultant clear orange-red solution wasextracted with ether (2×50 ml) and the aqueous phase was boiled on ahot-plate and concentrated to half the original volume (ca. 30 ml). Atthis point, the solution became turbid, and upon cooling a brown-orangesolid separated (810 mg). 750 mg (3.15 mmol) of this isomericmethoxy-hexanoresorcinones mixture was loaded on a silica gel columnequilibriated with dichloromethane. Fractions 6-7 contained pure (TLC)of the minor isomer. Evaporation of the solvent yielded 78 mg of whitesolid, which corresponds to example 107 (M.P. 121° C.). TLC(dichloromethane), R_(t)=0.33. ¹H NMR (CD₃OD, 200 MHz): δ5.90 (s, 2H),3.76 (s, 3H), 3.03 (t, 2H), 1.65 (t, 2H), 1.35 (m, 4H), 0.91 (t, 3H). MS(EI⁻): 167.1 (M⁺—C₅H₁₁, 100%), 238.2 (M⁻, 15%). Fractions 10-12 12 (4:1dichloromethane:ether was added at fraction 12) contained pure (TLC)major isomer. Removal of the solvents yielded 384 mg of grayish solid,which corresponds to example 108, (M.P. 109° C.). TLC (dichloromethane),R_(t)=0.10. ¹H NMR (CD₃OD, 200 MHz): δ5.94 (d, 1H), 5.87 (d, 1H), 3.84(s, 3H), 2.93 (t, 2H), 1.62 (t, 2H), 1.34 (m, 4H), 0.92 (m, 3H). MS(EI^(−):) 238.2 (M⁺, 39%), 167.1 (M⁺—C₅H₁₁, 100%). Example 107 wasalternatively prepared by esterification of 5-methoxy resorcinol withhexanoyl chloride and Fries rearrangement of the resultant mixture(procedure by Kay et at., U.S. Pat. No. 5,037,854).

Example 110

Procedure by Phloroglucinol (1.2 g, 9.5 mmol) was dissolved in anhydrousether (60 mL) at room temperature (26° C.) forming a clear solution.ZnCl₂ and acetonitrile (1.4 mL) were added to the clear solution. Theaddition of ZnCl₂ resulted in the formation of some sediment. Throughthe solution was passed a dry stream of freshly generated hydrogenchloride gas (1 mole total amount), which apparently caused the solutionto become cloudy. 5 minutes later, an orange oil formed and the solutionbrightened. After 2 additional minutes, the solution became orange andthe oil darkened. About 50 minutes later, when gas passage was complete,the system was closed and the oil solidified. Cold water (60 mL) wasadded to the orange crystals and solubility was achieved by vigorousstirring. The solution was extracted with ether (2×120 mL) andevaporated (to ˜30 mL). Upon cooling, airy, woolly colorless crystalsappeared (1.157 g, 73% yield). MS (EI⁺): 168.1 (M⁻, 46%), 153.1 (M⁺—CH₃,100%).

Example 51

Acetyl chloride (PCl₅-refluxed, quinoline-distilled, 290 μL, 4.08 mmol)was added to a clear yellow solution of cyanophenol (491 mg, 4.12 mmol)in Pyridine (5 mL). Heating and sedimentation were observed and thesolution was stirred overnight. Reverse phase HPLC showed completeconversion to a hydrophobic product (70% acetonitrile in H₃O, 1 mL/min,256 nm). The reaction contents were poured onto 5% HCl (50 mL) withstirring. A methylene chloride wash of the reaction flask (20 mL) wasadded to this mixture. After phase separation, the organic phase waswashed with sodium bicarbonate (50 mL) and aq. NaCl(sat. 50 mL), dried(Na₂SO₄), filtered and evaporated to yield an orange oil. 405 mg of thisoil (2.5 mmol) were heated on an oil bath (137° C.) and flushed withargon. AlCl₃ was added rapidly to a few portions while flushing. Thesolid brown-purple mass was cooled to room temperature, put on ice and5% HCl was added (10 mL) under ice. Ether was added (25 mL) and thephases were separated. The ethereal phase was extracted with 5% sodiumbicarbonate (25 mL). The orange aqueous phase contained some suspensionand was acidified by addition of 5% HCl (15 mL, until foaming ceased).An orange solid sedimented. Filtration and refiltration yielded 9 mg.HPLC (70% acetonitrile in H₂O, 1 mL/min, 256 nm): R_(t)=3.6′ (95-98%) ¹HNMR (CDCl₃, trace DMSO-d⁶, 200 MHz): δ13.00 (s, 1H, sharp), 8.01 (dd,1H, J₁=8.4 Hz, J₂=1.7 Hz), 7.79 (dd, 1H, J₁=7.5 Hz, J₂−1.9 Hz), 7.04 (t,1H, J=7.9 Hz), 2.70 (s, 3H). MS (EI⁻): 161.1 (M⁺, 10%), 44.0 (COH,100%).

II. Antiviral Assay—XTT Method

Antirhinoviral activity and cytotoxicity were determined by an XTTassay, as described previously for HIV-1 (Weislow et al., J Nat. Canc.Inst. 81(8):577-586, 1989) and for HRV-14 (Webber et al., J Med. Chem.5072-5082, 1996).

Specifically, each compound was assayed preferably according to thefollowing protocol. 24 hour-old Hl-Hela cells [ATCC] are plated in a96-well plate (˜30,000 cells/well, ˜60% confluence), washed with PBS(100 μL) and aspirated. Four 10-fold dilutions of virus (HRV 1A, 1B, 14or 16, obtained from ATCC) are prepared (in PBSA—0.1% BSA in PBS) andimmediately added (1 dilution per row, 20 μL per well) to the top halfof the plate (layout). The lower half of the plate is mock-infected bythe addition of 20 μL PBSA per well. The cells are incubated 1 hour atroom temperature (ca. 25° C.), and medium is introduced (DMEM+10% FCS:30 μL and Leibovitch medium (Biological Industries, Bet Haemek, Israel):50 μL.

A working solution of the assayed compound is prepared by dilution of anappropriate stock solution (in DMSO or ethanol) 40-fold in Leibovitchmedium, and 9 further dilutions are made in Leibavitch medium (no toxicor inhibitory effect observed in DMSO or ethanol controls). To each ofthe first ten columns is added a decreasing dilution of compound (50 μLper cell), followed by DMEM+10% FCS (30 μL per cell), followed byDMEM+10% FCS 30 μL per well). To each of the last two columns (untreatedvirus-infected and mock-infected cells) is added 50 μL Leibovitch mediumand 30 μL DMEM+10% FCS. A final volume of 100 μL per well (˜3% FCS) isthus obtained.

Format 1 −1.0 −1.1 −1.2 −1.3 −1.4 −1.5 −1.6 −1.7 −1.8 −1.9 VC-1 VC-1−2.0 −2.1 −2.2 −2.3 −2.4 −2.5 −2.6 −2.7 −2.8 −2.9 VC-2 VC-2 −3.0 −3.1−3.2 −3.3 −3.4 −3.5 −3.6 −3.7 −3.8 −3.9 VC-3 VC-3 −4.0 −4.1 −4.2 −4.3−4.4 −4.5 −4.6 −4.7 −4.8 −4.9 VC-4 VC-4 T.0 T.1 T.2 T.3 T.4 T.5 T.6 T.7T.8 T.9 C C T.0 T.1 T.2 T.3 T.4 T.5 T.6 T.7 T.8 T.9 C C T.0 T.1 T.2 T.3T.4 T.5 T.6 T.7 T.8 T.9 C C T.0 T.1 T.2 T.3 T.4 T.5 T.6 T.7 T.8 T.9 C C−1, −2, −3, −4: log 10 viral doses (−1, −2, −3, −4, respectively). .0,.1, .2, . . . wells treated with compound at 0, 1, 2, . . . 2-folddilutions, respectively T: Cytotoxicity (treated, mock infected). VC(dark-shaded area): virus control (virus-infected, untreated). C(light-shaded area): cell control (untreated mock-infected).

The plate is then incubated 2-3 days at 34° C. (5% CO₂), a mixture ofXTT (1 mg/mL in EMEM) and N-methylphenasonium methosulfate (PMS) isadded (50 μL). The plate is measured immediately (0-hour measurement)and following 3-5 hours of incubation at 37° C. (t-hour measurement) bya microplate reader (SLT-Lab Instruments-Austria, Model EAR-400) at awavelength of 450 nm (reference wavelength 620 nm). Corrected absorbancevalues (t-hour—0-hour measurements) are converted to a % cell-viabilityby arbitrary assignment of 100% viability to the average cell controlabsorbance (see layout). TC₅₀ values—defined as the concentration ofcompound that retards cell growth by 50%—are obtained by interpolationof or extrapolation from appropriate quadruple determinations (layout).IC₅₀ was defined as the concentration of compound that restores 50% ofthe viral-caused decrease in cell viability (relative to treateduninfected control). The decrease in viability resultant from virusaction was determined at each concentration of compound in relation tothe corresponding cytotoxicity. IC₅₀ values were determined by linearinterpolation (in some cases extrapolation).

Format 2 The plate layout was as follows: A₀ A₁ A₂ A₃ A₄ A₅ A₆ A₇ A₈ A₉VC VC A₀ A₁ A₂ A₃ A₄ A₅ A₆ A₇ A₈ A₉ VC VC A₀ A₁ A₂ A₃ A₄ A₅ A₆ A₇ A₈ A₉VC VC A₀ A₁ A₂ A₃ A₄ A₅ A₆ A₇ A₈ A₉ VC VC B₀ B₁ B₂ B₃ B₄ B₅ B₆ B₇ B₈ B₉CC CC B₀ B₁ B₂ B₃ B₄ B₅ B₆ B₇ B₈ B₉ CC CC B₀ B₁ B₂ B₃ B₄ B₅ B₆ B₇ B₈ B₉CC CC B₀ B₁ B₂ B₃ B₄ B₅ B₆ B₇ B₈ B₉ CC CC A/B = compound A or BA_(1,2,3) - 1^(st), 2^(nd), 3^(rd) 2-fold dilution of compound A CC -Cell control (uninfected cells only, no compound added) VC - Viruscontrol (infected cells, no compound added)

A single virus dilution was used per plate. Procedures prior to XTTaddition were either identical to format 1 or were performed accordingto the following protocol Compounds were titrated 2-fold in EMEM (in 50μL) and about 30,000 cells per well were added (in 30 μL EMEM with 10%FCS). The appropriate dilution of virus was then added (20 μL in EMEM).The final concentration of FCS was approximately 3%. XTT addition wassimilar to format 1. The incubation time was preferably 3-12 hours(usually 3-5 hours). The wavelength employed for plate reading wasgenerally as in format 1. In some cases, however, the measuringwavelength was 490 nm (reference wavelength 620 nm). Results wereobtained by 2-point linear interpolation (in some cases extrapolation)as in format 1. Results described herein correspond to VC viabilities of0-25% (75-100% loss of viability with virus relative to cell control).

Results

A) Results obtained by the XTT Assay in both formats are listed in thefollowing Tables (Compounds are from Aldrich Chem. Comp. unlessotherwise indicated). IC₅₀ and TC₅₀ values are micromolar.

Formula II Format 1 SA # X₁ X₂ R′ A A′ TC₅₀ IC₅₀ HRV 141 O O CH₃ CH₃COOCH₃ >1250 494 14 136 O O phenyl phenyl H 28 ± 2 6.3 14 131 O O2-hydroxybenzene phenyl H 47 ± 1 0.6 14 137 O O 5-chloro-2- phenyl H  5± 1 0.6 14 hydroxybenzene SA # X₁ X₂ R₁ A A′ IC₅₀ HRV 141 O O CH₃ CH₃COCH₃ 444 ± 313 14 520 ± 400 16 136 O O Phenyl phenyl H 13 ± 5  14 6 ± 316 131 O O 2-hydroxybenzene phenyl H 43 ± 9  1A 10 ± 2  14 0.5 ± 0.4 16142 O O 5-bromo-2-hydroxy- phenyl H 25 ± 12 1A benzene 59 ± 4  14 27 ±27 16 143 O O 5-methyl-2-hydroxy- phenyl H 8 ± 8 16 benzene 144 O O5-chloro-2-hydroxy-2- phenyl H 130 ± 40  14 methyl benzene 2.1 ± 1.6 16Formla III Formula III(i) Format 1 SA # Z′ Y′ R₃ Y TC₅₀ IC₅₀ HRV  12 HCl H H >2500 137 16  14 OMe H OMe H  780 ± 206 225 16  27 H F H H >1250444 14  28 H Br H H 1234 ± 610 6.2 14  29 H Cl H Cl 29 ± 8 4.8 14  30 HMe H NO2 67 ± 8 55 14  31 H Cl H NO2 12 ± 1 14 14  37 H OCH3 H H >1250146 14  38 H Br H Br 59 ± 4 26 14 110 OH H OH H 548 ± 27 192 14 425 16121 OCH2CONH2 H H H 425 ± 8  189 14 198 1A 132 OH H OH COOEt 29 ± 5 1714 13 1A 24 1B Compounds 121 and 132 synthesized in the Lab. Compound#14 by Signa Chemical SA # Z′ Y′ R₃ Y IC₅₀ HRV  6 H H H H 210 ± 170 14 7 H H OH H 810 ± 180 16  8 H OH H H 33 ± 3  1B 230 ± 170 14 35 ± 7  16141 ± 25  1A  9 OH H H H 110 ± 14  14 200 ± 110 16  12 H Cl H H 305 ±55  1B 150 ± 96  14 590 ± 300 16 290 ± 160 14  27 H F H H 47 ± 8  16  28H Br H H 91 ± 62 14 159 ± 44  16  29 H Cl H Cl 19 ± 8  1A 8 ± 5 1B 6 ± 214 6 ± 2 16  30 H Me H NO2 31 ± 12 14 29 ± 24 16  31 H Cl H NO2 80 ± 621A 11 ± 1  14 35 ± 26 16  37 H OCH3 H H 830 ± 330 1A 610 ± 160 1B 220 ±23  14 149 ± 148 16  38 H Br H Br 20 ± 7  1A 3.4 ± 1.5 1a 10 ± 2  14 6 ±2 16  41 H Cl Me H 160 ± 70  14 109 OH Cl H Cl 590 ± 320 14 121OCH2CONH2 H H H 120 ± 50  14 420 ± 140 16 138

OH Me 28 ± 5  14 Commercial source, except 109, 121 Formula III(ii)Format 1 SA # Z′ Y′ R₃ Y TC₅₀ IC₅₀ HRV  36 H OH H H 1720 ± 660  27 14Format 2 SA # Z′ Y′ R₃ Y IC₅₀ μM HRV  36 H OH H H 32 ± 4  14 44 ± 15 16Formula III(iii) Format 1 SA # Z′ Y′ R₃ Y TC₅₀ IC₅₀ HRV 116 H Cl OH Cl24 ± 6 13 14 21 16 118 OCH2CONH2 Cl OMe Cl 122 ± 6  380  14 81 1A 134OMe H OH Cl 48 ± 6 63 14 12 16 125 OMe Cl OH H 50 ± 5 45 14 60 16Syntheses of compounds by the Lab. SA # Z′ Y′ R₃ Y IC₅₀ μM HRV  15 OH ClOme Cl 5 ± 1 14 1.4 ± 0.8 16  16 OMe Cl OH Cl 28 ± 22 14 20 ± 13 16 107OH H Ome H 80 ± 40 14 34 ± 6  16 108 OMe H OH H 38 ± 6  14 41 ± 9  16114 H H OH H 40 ± 30 14 70 ± 60 16 116 H Cl OH Cl 50 ± 7  14 61 ± 37 16134 OMe H OH OH 122 ± 14  14 13 ± 6  16 All compounds synthesized by Labexcept SA-114 Formula III(iv) Format 1 SA # Z′ Y′ R₃ Y TC₅₀ IC₅₀ HRV  34OH H OH H 410 ± 30 94 14 Format 2 SA # Z′ Y′ R₃ Y IC₅₀ μM HRV  33 H Cl HCl 9 ± 1 1A 4 ± 1 1B 14 ± 3  14 6 ± 1 16  46 H NO₂ H H 170 ± 100 14  47H I H I 12 ± 2  14 Formula III(v) Format 1 SA # R′ Z′ R₃ TC₅₀ IC₅₀ HRV120 CH2Ph H H 271 ± 22 30 14 127 CH2CH2Ph H H 187 ± 18 23 14 128 n-C8H17H H 429 ± 42 152  14 Format 2 SA # R′ Z′ R₃ IC₅₀ μM HRV 120 CH2Ph H H 40± 33 14 30 ± 8  16 127 CH2CH2Ph H H 13 ± 3  1A 11 ± 1  1B 16 ± 4  14 15± 4  16 128 n-C8H17 H H 31 ± 6  14 4 ± 3 16 Synthesis of compounds bythe Lab. Formula III(vi) Format 1 SA # Y R₃ TC₅₀ IC₅₀ HRV 35 H H 180 ±10 62 14 Format 2 SA # Y R₃ IC₅₀ HRV  35 H H 630 ± 470 1A 240 ± 140 1B42 ± 20 14 38 ± 14 16 Formula III(vii) Format 1 SA # Z′ Y′ R₃ Y TC₅₀IC₅₀ HRV 1 H H OMe H 50 ± 10 16 14 Format 2 SA # Z′ Y′ R₃ Y IC₅₀ HRV  1H H OMe H 23 ± 20 14 165 ± 134 16 Formula III(viii) Format 1 SA # Z′ Y′R₃ Y TC₅₀ IC₅₀ HRV 137 H Cl H H 47 ± 10 6.2 1A 131 H H H H 5 ± 1 0.6 14Format 2 SA # Z′ Y′ R₃ Y IC₅₀ HRV 131 H H H H 43 ± 9  1A 10.5 ± 2.5  140.5 ± 0.4 16 142 H B H H 25 ± 12 1A 59 ± 4  14 27 ± 27 16 143 H CH₃ H H8 ± 8 16 144 H Cl CH₃ H 130 ± 40  14 2.1 ± 1.6 16 Formula III(ix) Format1 SA # Y′ R₃ Y TC₅₀ IC₅₀ HRV  3 H H H 10 ± 0.4 9 14 Format 2 SA # Y′ R₃Y IC₅₀ HRV  3 H H H 4 ± 2 14 72 ± 34 16 Formula III(x) Format 2 SA # Z′Y′ R₃ Y IC₅₀ HRV 147 OH H Me H 70 ± 50 16 Formula III(xi) Format 2 SA #Z′ Y′ R₃ Y R₁₁ IC₅₀ μM HRV  42 H H H H H 210 ± 70 1A 29 ± 1 1B 120 ± 4014  43 H Cl H H H 340 ± 70 16  40 ± 10 1B  30 ± 20 14  91 OH H OH H OH130 ± 14 1A  26 ± 15 1B  9 ± 4 14  50 ± 10 16 #43 Synthesized in lab #91Sigma Chem. Formula III(xii) Format 2 SA # Z′ Y′ R₃ Y R₁₁ R₁₂ IC₅₀ μMHRV  83 OH H OH H H H 250 ± 90  14  90 OH H OH H OH OH 5 ± 1 1A 7 ± 4 1B1.2 ± 0.5 14 7 ± 4 16 100 OH Cl OH Cl H H 27 ± 25 14 Sigma Chem. Comp.#10 Synthesis Formula IV Format 1 SA # X₂ Y R₃ Y′ R¹¹ R¹¹ R¹¹ R¹¹ TC₅₀IC₅₀ HRV 122 OH H H H OH OH H H 330 ± 30 104 1A (R¹¹ is used both for OHand for H clockwise from ketone) Format 2 SA # X₂ Y R₃ Y′ R¹¹ R¹¹ R¹¹R¹¹ IC₅₀ HRV 122 OH H H H OH OH H H 28 ± 5 1A 22 ± 5 1B 120 ± 40 14 28 ±6 16 Formula V Formula V(i) Format 2 SA # Z′ Y′ R₃ Y IC₅₀ HRV 149 H H HH 500 ± 400 1A 270 ± 90  1B 130 ± 50  14 29 ± 16 16 Formula V(ii) Format2 SA # Z′ Y′ R₃ Y IC⁵⁰ HRV 151 H Cl Me H 14 ± 6 14 Formula VI Format 2SA # Z′ Y′ Y IC₅₀ HRV  92 H H H 130 ± 50 16 B) Results relating toApopain modulation (inhibition and activation) were obtained byapplication of assay VI and are listed in the tables below (compoundsare from Aldrich Chem. Comp. unless otherwise indicated). Formula IIIFormula III(i) SA # Z′ Y′ R₃ Y IC₅₀ Activity  8 H OH H H 139 Inhibitor 9 OH H H H 133 Activator  27 H F H H 781 Inhibitor  30 H Me H NO2 235Inhibitor  37 H OCH3 H H 145 Inhibitor  51 H H H CN 146 Inhibitor 109 OHCl H Cl 179 Inhibitor 110 OH H OH H 241 Inhibitor 51, 109, 110 synthesisFormula III(iii) SA # Z′ Y′ R₃ Y IC₅₀ Activity  15 OH Cl Ome Cl  6Activator 107 OH H Ome H 213 Activator Synthesis Formula III(iv) SA # Z′Y′ R₃ Y IC₅₀ Activity  33 H Cl H Cl 59 Inhibitor  46 H NO₂ H H 64Inhibitor  47 H I H I 166  Activator Formula III(ix) SA # Y′ R₃ Y IC₅₀Activity  3 H H H <1 Inhibitor Formula xi (Z = OH, R₁ = CH₂CH₂Ph) SA #Z′ Y′ R₃ Y R₁₁ IC₅₀ Activity  91 OH H OH H OH 105 Inhibitor Sigma Chem

What is claimed is:
 1. A method for inhibiting the activity of apicornavirus 3C protease or a cysteine protease having an active sitestructure similar to a picornavirus 3C protease comprising exposing saidprotease to a chemical composition having an orthohydroxy keto aryl corestructure

wherein Z′ is —OCH₂CONH₂; and R₃, Y and Y′ are hydrogen.
 2. Acomposition comprising a compound having the following structure

wherein R₁ is methyl; and Y, R₃ and Y′ are hydrogen.